Interactions between ligands, such as peptides or small molecules, and cell surface proteins are crucial for numerous key processes in living organisms. Approximately one third of the mammalian genome encodes for membrane proteins. Activation and signalling of cell surface proteins are often involved in disease processes including malignant transformation. Identification of selective target membrane proteins can be used in the identification of biomarkers for early detection and prognosis of cancer but can also boost the discovery of new therapies. Moreover, mass spectrometry analysis has generated huge catalogues of possible bioactive peptides by the so-called peptidomics approaches. For many of these peptides that produce a biological effect it is currently not clear what their targets are or which molecular mechanisms they initiate. Indeed, for such orphan peptides the cell surface receptors or other protein partners generally remain unknown.
Today, the detection and visualisation of proteins, and more specifically cell surface proteins, is possible using a combination of primary and secondary antibodies. However, for the majority of cell surface proteins, no antibodies are commercially available and, even when available, the specificity (selectivity) and sensitivity (affinity) of commercially available polyclonal and monoclonal antibodies is often unsatisfactory for the detection of cell surface proteins. Furthermore, the use of antibodies is impossible for identifying unknown cell surface proteins.
Analysis of non-covalently bound cellular assemblies is, in particular for cell surface receptors, difficult for several reasons. Their hydrophobic nature and relatively low abundance precludes easy upscaling as they typically need to be in their natural environment to maintain binding properties. Activation of receptors by their ligands at the cell surface is based on the formation of a transient complex that, due to dynamic turnover, can be rapidly internalized and degraded. Immunoaffinity-based techniques for isolating ligand-receptor complexes from cell lysates are therefore rarely successful and can only be applied for high affinity interactions. Furthermore, interactomics techniques such as the yeast two-hybrid screen are unsuitable for the identification of extracellular protein-protein/peptide interactions. Elucidation of such interactions thus requires a technique compatible with living cells under physiological conditions.
In recent years, a whole range of bio-orthogonal chemistries was developed, allowing selective modification of biomolecules, in their natural environment. The introduced functional groups can react with a presented probe in an orthogonal way. Examples of such bio-orthogonal reactions include azide-alkyne cycloadditions, Staudinger ligation, and Diels Alder reactions. Despite the elegance and efficiency of these methods in labeling a wide variety of biomolecules, the need for modification of both binding partners with a specific unnatural reactive group represents a hurdle for general applicability.
Alternatively, photoaffinity crosslinking is based on the introduction of a photoreactive group which is able to form a crosslink with an unmodified natural binding partner upon activation with UV light. This requirement for an activation step limits the applicability of photo-crosslinking in complex biological settings such as living cells. Benzophenones, aryl azides and diazirines are among the most widely used groups. Although previously applied in the characterization of ligand-receptor complexes, these chemistries bear several disadvantages. Phototoxicity needs to be strictly monitored. Furthermore, the formation of highly reactive intermediates reduces selectivity of crosslinking. Therefore, experiments are generally carried out with cell lysates or, when working with living cells, in cold buffers. Benzophenones are typically bulky groups, which may negatively influence biological activity of the used probes. Aryl azides are much smaller, but the short-wave UV-light needed for their activation (<300 nm), is known to cause damage to the biological environment. Finally, diazirines are more stable, are excited at higher wavelengths, but their synthesis is quite tedious.
Recently, a crosslinking technology was developed based on a furan moiety. Furan represents a latent reactive moiety that needs primary oxidative activation to allow covalent bond formation with nucleophilic sites in its proximity. Furan, when incorporated in oligonucleotides, can be activated by oxidation using N-bromosuccinimide (NBS) (Halila et al., 2005, Chem. Commun. (Camb), 936-8) or singlet oxygen (Op de Beeck and Madder, 2012, J. Am. Chem. Soc., 134, 10737-40).
WO2012/085279 describes a method for crosslinking peptides comprising a furan moiety with second peptides. The method requires the addition of an activation signal to oxidize the furan moiety. The example section of WO2012/085279 illustrates that crosslinking furan-StrepTagII peptides with streptavidin required the addition of NBS as an activation signal to oxidize the furan moiety.
Although furan is commercially available, its use in cell-based assays has always been avoided in view of its toxicity and carcinogenicity. In the liver, cytochrome P450 catalyses oxidation of furan to a reactive aldehyde, which subsequently reacts with sulfhydryl and amine groups (Chen et al., 1997, Chem. Res. Toxicol., 10, 866-74).
In view of the above, there remains a need in the art to provide further and/or improved methods for detecting and/or identifying cell surface proteins or ligands specifically binding to cell surface proteins.